Project Summary Despite the concerns of possible estrogenic chemical leachates in coffee extract solutions made with capsule coffee, market share for these products has been rapidly growing during the past several years. The capsule coffee brands claim that the capsules are bisphenol A (BPA)-free. However, a series of recent studies by our research team demonstrated that BPA-free does not mean that they are free of estrogenic chemicals (ECs). In addition, although the exposure levels to these chemicals may be very low, the safety and health implications of long-term habitual drinking of capsule coffee remain to be investigated. Nevertheless, no study has been conducted to assess the exposure levels of these chemicals through the use of capsule coffee products. Furthermore, studies on their effects on human health are lacking. Therefore, to assure the safety or acceptable risk associated with the use of capsule coffee, it is of importance to conduct studies that systematically evaluate the health risk of the consumption of capsule coffees based on comprehensive residual chemical analysis, estrogenicity cell tests, and human risk exposure studies. This study aims to assess the risk of exposure to ECs through the use of coffee capsules. Our central hypothesis for the proposed work is that consumption of capsule coffee increases the risk of exposure to ECs. To test this hypothesis we will conduct comprehensive chemical profiling of capsule coffee extracts using ultra- performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) to detect unknown potential ECs as well as ECs commonly found in foods and food containers including BPA, BPF, BPS, Tritan copolyester components (dimethyl terephthalate, 1,4-cyclohexanedimethanol, and 2,2,4,4-tetramethyl-1,3- cyclobutanediol), nonylphenol (NP), and phthalates (di-2-ethylhexyl phthalate, DEHP, and di-n-butyl phthalate, DBP) (Aim 1) and test the estrogenicity of coffee extracts by MCF-7 and BG1Luc cell assays (Aim 2). Additionally, we will conduct a randomized crossover study with capsule coffee and pour-over coffee (positive control) extracts to evaluate the impact of capsule coffee consumption on exposure to ECs by comparing urinary EC concentrations after ingestion of coffee extract solutions in healthy volunteers (Aim 3). The proposed study will identify and quantitate the levels of ECs in capsule coffee solutions and evaluate the risk of exposure to these chemicals via capsule coffee consumption. Findings from this study will provide important and time-sensitive information to comprehensively understand human exposure to ECs through capsule coffee consumption and also provide scientific background to establish a strategy to lower the exposure levels to these chemicals through dietary and lifestyle modifications.